The availability of a large number of biological reagents, such as hundreds of thousands of cloned DNA sequences, numerous antibodies and recombinant proteins, millions of compounds obtained through combinatory chemical synthesis, has promoted the development of technologies that make use of these reagents in biological research, clinical diagnostics and drug development. Special position-addressable arrays of biological reagents have been designed, in which each of the reagents is placed at a pre-defined position so that it can be identified later by the position. For example, in a DNA array, a large number of cDNA or oligos are immobilized, each at a pre-defined position and can be identified later by that position. DNA arrays are used in large-scale hybridization assays for applications such as monitoring gene expressions (Schena et al., 1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457-460). Arrays of DNA clones in expression vectors are also used to express their encoded proteins in mammalian cells (Ziauddin and Sabatini, 2001, Nature 411, 107-110).
In a protein array, many proteins are immobilized on a support, each at a predetermined position so that it can be identified subsequently by this unique position. Two types of protein arrays are particularly useful: antibody arrays and recombinant protein arrays, which contain a plurality of antibodies and a plurality of recombinant proteins, respectively. Because antibody arrays are capable of binding cellular proteins, they are particularly useful in revealing protein in vivo activities. Therefore, the technology makes it possible to study the properties of a large number of cellular proteins in a single assay. Specifically, antibody arrays have been applied in studying in vivo protein-protein interactions, protein posttranslational modifications and protein expression patterns (U.S. Pat. No. 6,197,599).
In addition, arrays of cells, tissues, lipids, polymers, drugs and other chemical substances can be fabricated for large scale screening assays in medical diagnostics, drug discovery, molecular biology, immunology and toxicology (Kononen, et al., Nature Medicine, 4:844-7, 1998).
Proteins are important component of cells and they are the real players in various cellular processes; and they are the targets of most drugs. The entire human genome encodes about 40,000 proteins. Although a given cell may contain the DNA encoding all the proteins, it usually only expresses a fraction of them. A cell line usually expresses about 10,000 proteins and an even higher number is expressed in tissues. The protein expression pattern of a cell determines its shape and function; and abnormal protein expressions cause diseases. Therefore, one major task of proteomics is to identify the proteins expressed in a given source.
A protein (with an identical primary amino acid sequence) may be present in different forms in the cells largely due to posttranslational modifications. Since, in many cases, only special posttranslationally modified proteins are activated and directly involved in a cellular process, the detection of the presence of these activated proteins in the cells can provide valuable information on that cellular process.
There are many different protein posttranslational modifications such as phosphorylation, glycosylation, and ubiquitination. And they play important roles in regulating protein activities. Phosphorylation in either serine, threonine or tyrosine residues is an important mechanism in signal transduction. Aberrant protein phosphorylation contributes to many human diseases. Among the methods of detecting protein phosphorylations, metabolic labeling of cells with radioisotopes and immuno-detection with antibodies against phosphoproteins is most commonly used. However, these methods are usually only applicable to the analysis of one or a few proteins at a time. Although antibodies specific for phosphorylated amino acids, such as PY20 and 4G10, can reveal multiple phosphorylated proteins, they alone are unable to identify individual phosphorylated proteins. New methods for simultaneously detecting the presence of multiple phosphorylated proteins or other modified proteins are highly desirable for signal transduction studies and clinical diagnosis.
Quantification of protein expressions has applications in a variety of fields including biomedical research, disease diagnosis, identification of therapeutic markers and targets, and in profiling cellular responses to toxins and pharmaceuticals. In basic biomedical research, it is usually desirable to know what proteins are expressed in specific cells or under specific conditions. And by comparing the protein expression profiles between different cell types, it is possible to identify those proteins whose expressions and activations characterize a particular cell type. In many signal transduction pathways, certain proteins are specifically activated; and the detection of these active proteins, e.g., phosphorylated proteins, may provide important information on the activations of specific signal transduction pathways.
Many diseases alter protein expressions and in many cases abnormal protein expressions are the causes of the diseases. Therefore, determination of protein expression profiles and comparison of the expression profiles between normal and abnormal biological samples are useful for understanding disease mechanisms. Detecting the presence of proteins is also useful in clinical diagnostics. For example, examination of the presence of several viral proteins instead of just one in a blood sample is a more reliable diagnostic method for viral infections. Profiling proteins will be invaluable in distinguishing normal cells from early-stage cancers and also from malignant, metastatic cancer cells that are the real killers. In addition, protein expression profiling is useful in key areas of drug development, such as in drug target selection, toxicology and the identification of surrogate markers of drug response.
It has long been the goal of molecular biologists to develop technologies that can quantify, in a reliable and reproducible manner, the expression level of every individual protein and the different forms of each protein in a biological sample. However, this has turned out to be extremely difficult to achieve. Traditionally, the expression of one or a small number of proteins can be detected by immunological methods, such as western blotting and Enzyme-Linked Immunosorbent Assay (ELISA). Two-dimensional gel electrophoresis can be used to analyze the proteins expressed in a sample. However, it requires complicated procedures and it is necessary to determine the identities of the proteins displayed on the two-dimensional gel, which is difficult to achieve for most proteins. Recently, protein arrays are applied in studying protein expression patterns. In one strategy (U.S. Pat. No. 6,197,599; Haab, et al., Genome Biol. 2, research0004.1-0004.13, 2001), an antibody array is incubated with a protein sample and after incubation and washing, proteins specifically bound to their respective antibodies on the array are detected.
Immunochemical staining is a versatile technique in determining both the presence and localization of an antigen (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988), which information is of immense value in biomedical research and clinical medicine. Most of the current methods, which employ the steps of incubating cells with an antibody solution, only allow cell staining with one or a few antibodies at a time. These methods are not suitable for applications in which the expressions and sub-cellular localizations of a large number of different proteins need to be examined. Therefore, a new method that is capable of staining cells with a large number of different antibodies is needed for such purposes.